The determination of the amino terminal amino acid sequences of proteins and peptides is an essential analysis for their identification or confirmation.
However, 60% or more of the soluble proteins derived from eukaryote reportedly have the .alpha.-amino group at their amino terminal blocked by either an acetyl group or another protecting group; and in the Edman degradation method, a well-established commonly used method for. analyzing an amino acid sequence from the amino terminal, proteins and peptides of which amino terminal is blocked by a protecting group cannot be analyzed.
As the protecting groups blocking the amino terminal of proteins and peptides, formyl group, acetyl group, myristoyl group, pyroglutamyl group, dimethyl group, glucuronyl group, glycosyl group and trimethyl group have been reported. As the method for analyzing an amino terminal amino acid sequence for such blocked proteins, there can be employed the Edman degradation method applied after removing a protecting group.
The removal of protecting groups includes a method using an enzyme and a chemical method. However, an enzymatic method has a drawback of a lack of versatility, since different enzymes are used depending on the kinds of protecting groups to be removed. For example, acylamino acid releasing enzyme is used in the case of acetyl groups, and pyroglutamyl peptidase in the case of pyroglutamyl groups. Also, as to chemical method, no versatile methods have been developed to date. In addition, because there is no known method of identifying the protecting group when the amino terminal is blocked thereby, when the protecting group is actually removed, there are no other alternatives except that a number of methods for removing protecting group depending on respective protecting groups are attempted one by one. Furthermore, since there is no effective method for removing a protecting group available for the proteins and peptides blocked with myristoyl group, it is impossible to determine their amino acid sequences from the amino terminal.
Incidentally, as the peptidases isolated from Pyrococcus furiosus, the peptidases acting on an amino terminal portion of a peptide, there have been known an aminopeptidase (Japanese Patent Laid-Open No. Hei 6-319566), a pyroglutamyl peptidase (Japanese Patent Laid-Open No. Hei 7-298881), and a methionine aminopeptidase (Japanese Patent Laid-Open No. Hei 8-9979). Among these peptidases, the pyroglutamyl peptidase possesses an activity for releasing of the amino terminal pyroglutamyl group, but does not act on other protecting groups, e.g., acetyl groups. In addition, the other two kinds of enzymes both cannot act on amino terminal blocked by protecting groups.
As described above, the existing method for analyzing amino terminal amino acid sequence has the drawback of a lack of versatility when applied to proteins and peptides of which amino terminal is blocked by protecting groups.